Expression And Function Of ?-Tubulin And H3S10ph During Porcine Oocyte-to-Embryo Transition

In this study,the expression and function of a-Tubulin and H3S10ph during porcine oocyte-to-embryo transition were studied by means of indirect immunofluorescence,laser confocal microscopy,immunoblotting and specific inhibitor treatment.First,the dynamic distribution of cytoskeleton(tubulin and microfilament protein)in oocyte maturation and embryo first mitosis was examined,and the function of tubulin was studied in chromosome migration and separation by colchicine treatment.In order to study the effect of H3S10ph on chromosome condensation during the first mitosis of porcine embryos,we first used protein immunoblotting and indirect immunofluorescence to detect the expression and subcellular localization of H3S10ph in the first mitosis of porcine embryos.On this basis,H3S10ph upstream protein Aurora-B specific inhibitor to study the function of H3S10ph on first cleavage cell cycle and chromosome condensation during the first mitosis in porcine embryos.Looking forward to put further insight in possible regulation of H3S10ph during first mitotic division in porcine embryos.The main experimental results are as follows:Experiment 1 Dynamic distribution of cytoskeleton during during porcine oocyte-to-embryo transitionImmunofluorescence staining showed that ?-Tubulin began to be assembled after GVBD,and a typical spindle structure was formed at MI,?-Tubulin was distributed in the spindle between the two groups of chromosomes at A?,and was formed a typical spindle at M? again near the chromosome;F-actin was enriched in the extracellular cortical region after GVBD,enriched at cortical region and formed actin cap where adjacent the spindle at MI and M?.During the first mitotic division in porcine embryo,microtubules were found throughout the cytoplasm at the prophase stage.At the prometaphase stage,microtubules were detected in association with chromatin mass and encompassed the chromatin.Microtubules formed a symmetric and barrelshaped structure spindle at metaphase stage.During anaphase,microtubules were found in the mitosis spindle.At cytokinesis,microtubules were detected in the mitosis midbody.Microflaments were observed as a relatively thick and uniform area around the cell cortex and were also distributed throughout the cytoplasm of embryos at different stages during the mitosis.Microflaments were particularly condensed at the cleavage groove during cytokinesis.On the basis of this,oocytes were treated after MI with colchicine,the majority of oocyte spindles were depolymerized,the chromosomes were disordered,and pbI discharge rate was decreased significantly;but after colchicine was removed,a-Tubulin can be re-formed spindle structure at MI,pbI excretion rate was recoveried when extended the culture time.The results of this study were shown that the dynamic changes of cytoskeleton in porcine oocyte maturation and the first mitosis of embryos are characterized by phylogenetic distribution.The subcellular localization of a-Tubulin is closely related to chromosome migration and its polarization to the poles.colchicine can destroy the spindle structure in the porcine oocyte during MI,and inhibit pbI discharge,and the of colchicion effects on the spindle was reversible.Experiment 2 H3S10ph levels and subcellular localization in porcine embryos during the frst mitotic divisionThe results of immunoblotting showed that H3S10ph was expressed in the first mitotic period of porcine embryos,and H3S10ph levels were maximal during prometaphase and reduced during meta-anaphase and cytokinesis.Immunofluorescence assay showed that,A dynamic subcellular localization pattern of H3S10ph during the frst embryo mitosis is presented in porcine embryos during the first mitotic division.H3S10ph was distributed throughout the cytoplasm of embryos during prophase and then concentrated on chromatin during prometaphase,metaphase and anaphase.When embryos progressed to cytokinesis,H3S10ph was distributed throughout the cytoplasm of the embryo.the Aurora-B inhibitor caused the failure of the first mitotic division in porcine embryos.The above results indicate that H3S10ph is continuously expressed during the first mitotic division in porcine embryos and has close temporal and spatial correlation with chromosome dynamics.H3S10ph with specific inhibitor AZD-1152 treatment resulted in the failure of the porcine embryo during the first mitotic division,suggesting that H3S10ph is a key regulator during the frst mitotic division in porcine embryos.Experiment 3 Regulatory Mechanism of H3S10ph during porcine embryo first mitotic divisionIn this study,porcine embryonic cells were treated by using H3S10ph upstream regulatory protein Aurora B specific inhibitor AZD-11152 to study the regulatory mechanism of H3S10ph during the frst mitotic division in porcine embryos.The results showed that the proportion of embryos in the cytoplasmic phase was significantly decreased after 48 h with the inhibitor AZD-1152.compared with the normal group,the proportion of embryonic cells in the prophase was significantly increased.The embryos were cultured for 20 h,the chromosome of AZD-1152 treated group was not condensed compared with the control group,and the level of H3S10ph was significantly decreased.The above results indicate that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function on chromosome condensation,which further affects the cell cycle and frst mitotic division in porcine embryos.